The present invention relates to an inactivated viral vaccine. More specifically, the invention relates to a method for preparing dengue virus vaccine using psoralen inactivated viral particles.
Dengue viruses consist of four distinct but closely related single-stranded, enveloped RNA viruses of the genus Flaviviridae. Geographically widespread in tropical and subtropical regions, dengue viruses are transmitted to humans primarily through the bite of the Aedes aegypti mosquito. Between 50-100 millions human infections occur annually due to dengue, making it the world's most widespread arboviral disease. Clinical manifestations vary among hosts, with presentations that include asymptomatic infections, nonspecific febrile syndromes, the more severe (classic) dengue fever, and finally the life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). There are an estimated 250,000 cases per year of DHF and DSS worldwide. Most affected patients are young children, who also make up the majority of the approximately 25,000 estimated annual deaths due to dengue infection1.
Therapeutic options for dengue infection currently are limited to supportive care. No specific therapy for dengue fever exists, although treatment such as aggressive fluid management with crystalloid solutions has been shown to be effective2. However, immunomodulation with corticosteroids3 or intravenous immunoglobulin4 has not demonstrated any benefit in human trials.
Primary dengue infection leads to short-lived heterologous immunity to all four dengue serotypes5. However, lasting immunity persists only to the infecting serotype, leaving previously-infected persons susceptible to the other three serotypes of the virus. Many endemic regions of the world have multiple circulating serotypes at any given time, limiting the protection gained from a prior infection. Furthermore, prior dengue infection is a major risk factor for the subsequent development of DHF and DSS following re-infection with a heterotypic dengue serotype. Although the molecular and immunologic mechanisms for DHF are not completely understood, it is believed that antibodies to the primary infecting serotype interact with the envelope (E) protein on the surface of a heterotypic dengue serotype, which leads to an increased uptake of virus into susceptible host cells through an immunoglobulin constant chain receptor (FcR)-dependent mechanism6. These infected cells permit dengue virus to replicate in greater quantities, causing increased viremia and disease severity. Homotypic antibodies to the infecting serotype plays a major role in the prevention of recurrent infection, although it is uncertain to what extent humoral factors are responsible for this immunity as compared with cell-mediated immunity30, 31. Heterotypic antibodies to a prior infecting dengue serotype appear to contribute to the subsequent development of DHF, with these heterotypic antibodies enhancing the ability of dengue viruses to infect cells via binding to the immunoglobulin FcR32, 33. Additionally, low-affinity cell-mediated responses to dengue re-infection may also lead to excessive cytokine release, contributing to the vascular leak that is the hallmark of DHF7.
As a result, dengue vaccine development has been hindered by the competing interests to develop a vaccine that provides lasting and uniform protection against all four dengue serotypes, and the need to avoid predisposing the vaccine recipients to an increased risk of DHF and DSS. Vaccine candidates currently in development include a recombinant live-attenuated viral vaccine, DNA vaccines encoding for dengue viral epitopes, and a vaccine using a recombinant YF17D (vaccine strain) yellow fever virus with a modified genome that encodes for dengue-specific antigen8. Phase 1 and 2 human trials have been completed for some of these candidates9, 10, 11, 12, but none is presently ready for commercial release. These products show promise but may require boosting, either with multiple vaccine doses or with a separate agent.
A successful dengue vaccine will need to produce high-affinity and enduring antibodies to all four serotypes, possibly by means of periodic booster doses. There are promising vaccine candidates that utilize individual dengue epitopes (such as domain III of the dengue envelope (E) protein). An inactivated viral vaccine, by contrast, has the potential to present these epitopes to the host immune system in their native confirmation. In addition, cell-mediated immunity is generally lacking from inactivated vaccines due to the absence of viral replication. The exact contribution of cell-mediated immunity to the development of severe dengue is unclear. T-lymphocytes may assist with containment of recurrent homotypic infections through the elimination of excess virus not controlled adequately by antibody, but there is evidence that low-affinity T-cell responses to heterotypic infection contributes to DHF by excessive cytokine production (such as TNF-alpha)30, 34, 35. Prior T-cell immunity does not seem to be a necessary step in the pathogenesis of DHF, however, as demonstrated by the occurrence of DHF in infants with residual maternal anti-dengue antibody but no personal history of dengue infection36. Therefore, an inactivated vaccine may overcome the problems associated with excessive cytokine release.
Psoralen (also called psoralene) is the parent compound in a family of natural products known as furocoumarins. Psoralens are photoreactive compounds that are freely permeable in phospholipid membranes and intercalate between double-stranded nucleic acids. Following exposure to long wave ultraviolet radiation (UVA) with wavelength of 300-400 nm, the intercalated psoralen covalently crosslink complementary pyrimidine residues, leading to viral inactivation through inhibition of genome replication. Psoralen interaction with viral nucleic acids leaves immunogenic surface epitopes intact13, raising the possibility that a psoralen-inactivated virus may serve as a vaccine candidate. This photo-crosslinking property of psoralens has been exploited to inactivate microorganisms in the blood supply14, 15, for treatment of skin disorders16, to inactivate viral pathogens prior to organ transplantation17, and for inactivation of viruses for potential vaccines18, 19, 20.
Psoralen-inactivated viruses should, in theory, retain their three-dimensional structure, permitting the development of antibodies to multiple epitopes that may participate in immunity. Psoralens are freely permeable through lipid bilayers and do not appear to interact with proteins. Additionally, they only induce cross-linking of pyrimidines following UV exposure24, 25. This feature of psoralens has made them attractive in transfusion medicine for pathogen inactivation, wherein they damage the nucleic acid of pathogenic contaminants without disrupting the donor-derived erythrocytes, platelets, and coagulation factors themselves14, 15, 26.
Psoralens also have a good safety record in humans. There is increasing experience with the use of psoralens in blood banking, particularly the use of amotosalen as an alternative to traditional leukoreduction methods for the prevention of CMV transmission14. The oral and topical use of psoralens in the treatment of psoriasis has been associated with photosensitivity, contact dermatitis, and DNA damage in histologic specimens from treated tissue27, 28, 29. These adverse reactions are due in part to the direct exposure of human skin to UVA and psoralen, and do not seem likely to be of concern following vaccine preparation, assuming adequate purification of the inactivated virus preparation. Good safety record and photo-crosslinking property of psoralens lend it to possible use in inactivating virus vaccines.
U.S. Pat. Nos. 4,124,598 and 4,196,281 to Hearst et al. suggest the use of psoralen derivatives to inactivate RNA viruses, but include no discussion of the suitability of using the inactivated viruses for vaccines. U.S. Pat. No. 4,169,204 to Hearst et al. suggest that psoralens may provide a mean for inactivating viruses for the purpose of vaccine production but presents no experimental support for this proposition. European patent application 0 066 886 by Kronenberg teaches the use of psoralen inactivated cells, such as virus-infected mammalian cells, for use as immunological reagents and vaccines. Hanson (1983) reported studies suggesting that oxidative photoreactions between psoralens and proteins may occur37. However, it also has been recognized that inactivation of viruses by exposure to ultraviolet radiation in the presence of furocoumarin compounds can degrade the antigenic structure of the viral particles. While such degradation can be limited by employing less rigorous inactivation conditions, certain recalcitrant viruses require relatively harsh inactivation conditions in order to assure that all residual infectivity is eliminated. Thus, the inactivation conditions required to eliminate substantially all infectivity in such recalcitrant viruses can also degrade the viral particle so it is unsuitable for use as the immunogenic substance in a vaccine. Even if the degradation is not so complete, partial degradation of the antigenic characteristics may render the vaccine less effective than would be desirable. That is, the vaccine may require higher concentrations of the inactivated viral particles in each inoculation, and/or the vaccination program may require additional inoculations in order to achieve immunity.
U.S. Pat. No. 4,693,981, by Wiesehahn et al. disclosed an improved method for preparing inactivated viral vaccine without substantially degrading its antigenic characteristics. Wiesehahn et al prepare a selected group of viruses for vaccine uses by exposing the virus to a preselected concentration of an inactivating furocoumarin and a preselected intensity of ultraviolet radiation in the presence of inert gases or oxygen scavengers. However, this method calls for long period (2-60 hours) of high intense UVA exposure (0.1 m W/cm2 to about 5 W/cm2), which must be carried out in absence of oxygen or oxygenized species to preserve the antigenic characteristics of the virus. The absence of oxygen and oxygenized species are maintained through additional processes, such as removing the oxygenized species from the inactivation medium prior to irradiation through flushing with non-oxidizing gas and adding oxygen scavengers to the medium. The patent contains no discussion of the suitability of this method in preparing inactivating dengue viruses or Flaviviridae virus vaccines.
Although psoralen is known to be effective in inactivating viruses, its suitability for preparing an inactivating arboviral vaccine or more specifically a dengue viral vaccine is still unclear. The optimal inactivation conditions for such vaccine preparation, including the length of UVA exposure, the UV intensity, the concentration and selection of psoralen, are left to be experimentally determined, which is the objective of this study.